Modified Nucleic Acid Probes

ABSTRACT

Oligonucleotide analogue arrays attached to solid substrates and methods related to the use thereof are provided. The oligonucleotide analogues hybridize to nucleic acids with either higher or lower specificity than corresponding unmodified oligonucleotides. Target nucleic acids which comprise nucleotide analogues are bound to oligonucleotide and oligonucleotide analogue arrays.

CROSS-REFERENCE TO RELATED APPLICATION

This application is a continuation of U.S. Ser. No. 12/124,061 filed May20, 2008, now U.S. Pat. No. 7,794,943, which is a continuation of U.S.Ser. No. 10/418,414 filed Apr. 18, 2003, now U.S. Pat. No. 7,375,198,which is a continuation of U.S. Ser. No. 09/608,691 filed Jun. 29, 2000(now abandoned), which is a continuation of Ser. No. 08/630,427 filedApr. 3, 1996, now U.S. Pat. No. 6,156,501, which is acontinuation-in-part of U.S. Ser. No. 08/440,742 filed May 10, 1995,(now abandoned), which is a continuation-in-part of PCT application(designating the United States) SN PCT/US94/12305 filed Oct. 26, 1994,which is a continuation-in-part of U.S. Ser. No. 08/284,064 filed Aug.2, 1994, which is a continuation-in-part of U.S. Ser. No. 08/143,312filed Oct. 26, 1993, (now abandoned), each of which is incorporatedherein by reference in its entirety for all purposes.

FIELD OF THE INVENTION

The present invention provides probes comprised of nucleotide analoguesimmobilized in arrays on solid substrates for analyzing molecularinteractions of biological interest, and target nucleic acids comprisedof nucleotide analogues. The invention therefore relates to themolecular interaction of polymers immobilized on solid substratesincluding related chemistry, biology, and medical diagnostic uses.

BACKGROUND OF THE INVENTION

The development of very large scale immobilized polymer synthesis(VLSIPS™) technology provides pioneering methods for arranging largenumbers of oligonucleotide probes in very small arrays. See, U.S.application Ser. No. 07/805,727 and PCT patent publication Nos. WO90/15070 and 92/10092, each of which is incorporated herein by referencefor all purposes. U.S. patent application Ser. No. 07/082,937, filedJun. 25, 1993, and incorporated herein for all purposes, describesmethods for making arrays of oligonucleotide probes that are used, e.g.,to determine the complete sequence of a target nucleic acid and/or todetect the presence of a nucleic acid with a specified sequence.

VLSIPS™ technology provides an efficient means for large scaleproduction of miniaturized oligonucleotide arrays for sequencing byhybridization (SBH), diagnostic testing for inherited or somaticallyacquired genetic diseases, and forensic analysis. Other applicationsinclude determination of sequence specificity of nucleic acids,protein-nucleic acid complexes and other polymer-polymer interactions.

SUMMARY OF THE INVENTION

The present invention provides arrays of oligonucleotide analoguesattached to solid substrates. Oligonucleotide analogues have differenthybridization properties than oligonucleotides based upon naturallyoccurring nucleotides. By incorporating oligonucleotide analogues intothe arrays of the invention, hybridization to a target nucleic acid isoptimized.

The oligonucleotide analogue arrays have virtually any number ofdifferent members, determined largely by the number or variety ofcompounds to be screened against the array in a given application. Inone group of embodiments, the array has from 10 up to 100oligonucleotide analogue members. In other groups of embodiments, thearrays have between 100 and 10,000 members, and in yet other embodimentsthe arrays have between 10,000 and 1,000,0000 members. In preferredembodiments, the array will have a density of more than 100 members atknown locations per cm², or more preferably, more than 1000 members percm². In some embodiments, the arrays have a density of more than 10,000members per cm².

The solid substrate upon which the array is constructed includes anymaterial upon which oligonucleotide analogues are attached in a definedrelationship to one another, such as beads, arrays, and slides.Especially preferred oligonucleotide analogues of the array are betweenabout 5 and about 20 nucleotides, nucleotide analogues or a mixturethereof in length.

In one group of embodiments, nucleoside analogues incorporated into theoligonucleotide analogues of the array will have the chemical formula:

wherein R¹ and R² are independently selected from the group consistingof hydrogen, methyl, hydroxy, alkoxy (e.g., methoxy, ethoxy, propoxy,allyloxy, and propargyloxy), alkylthio, halogen (Fluorine, Chlorine, andBromine), cyano, and azido, and wherein Y is a heterocyclic moiety,e.g., a base selected from the group consisting of purines, purineanalogues, pyrimidines, pyrimidine analogues, universal bases (e.g.,5-nitroindole) or other groups or ring systems capable of forming one ormore hydrogen bonds with corresponding moieties on alternate strandswithin a double- or triple-stranded nucleic acid or nucleic acidanalogue, or other groups or ring systems capable of formingnearest-neighbor base-stacking interactions within a double- ortriple-stranded complex. In other embodiments, the oligonucleotideanalogues are not constructed from nucleosides, but are capable ofbinding to nucleic acids in solution due to structural similaritiesbetween the oligonucleotide analogue and a naturally occurring nucleicacid. An example of such an oligonucleotide analogue is a peptidenucleic acid or polyamide nucleic acid in which bases which hydrogenbond to a nucleic acid are attached to a polyamide backbone.

The present invention also provides target nucleic acids hybridized tooligonucleotide arrays. In the target nucleic acids of the invention,nucleotide analogues are incorporated into the target nucleic acid,altering the hybridization properties of the target nucleic acid to anarray of oligonucleotide probes. Typically, the oligonucleotide probearrays also comprise nucleotide analogues.

The target nucleic acids are typically synthesized by providing anucleotide analogue as a reagent during the enzymatic copying of anucleic acid. For instance, nucleotide analogues are incorporated intopolynucleic acid analogues using taq polymerase in a PCR reaction. Thus,a nucleic acid containing a sequence to be analyzed is typicallyamplified in a PCR or RNA amplification procedure with nucleotideanalogues, and the resulting target nucleic acid analogue amplicon ishybridized to a nucleic acid analogue array.

Oligonucleotide analogue arrays and target nucleic acids are optionallycomposed of oligonucleotide analogues which are resistant to hydrolysisor degradation by nuclease enzymes such as RNAase A. This has theadvantage of providing the array or target nucleic acid with greaterlongevity by rendering it resistant to enzymatic degradation. Forexample, analogues comprising 2′-O-methyloligoribonucleotides areresistant to RNAase A.

Oligonucleotide analogue arrays are optionally arranged into librariesfor screening compounds for desired characteristics, such as the abilityto bind a specified oligonucleotide analogue, or oligonucleotideanalogue-containing structure. The libraries also includeoligonucleotide analogue members which form conformationally-restrictedprobes, such as unimolecular double-stranded probes or unimoleculardouble-stranded probes which present a third chemical structure ofinterest. For instance, the array of oligonucleotide analoguesoptionally include a plurality of different members, each member havingthe formula: Y-L¹-X¹-L²-X², wherein Y is a solid substrate, X¹ and X²are complementary oligonucleotides containing at least one nucleotideanalogue, L¹ is a spacer, and L² is a linking group having sufficientlength such that X¹ and X² form a double-stranded oligonucleotide. Anarray of such members comprise a library of unimolecular double-strandedoligonucleotide analogues. In another embodiment, the members of thearray of oligonucleotide are arranged to present a moiety of interestwithin the oligonucleotide analogue probes of the array. For instance,the arrays are optionally conformationally restricted, having theformula —X¹¹—Z—X¹², wherein X¹¹ and X¹² are complementaryoligonucleotides or oligonucleotide analogues and Z is a chemicalstructure comprising the binding site of interest.

Oligonucleotide analogue arrays are synthesized on a solid substrate bya variety of methods, including light-directed chemical coupling, andselectively flowing synthetic reagents over portions of the solidsubstrate. The solid substrate is prepared for synthesis or attachmentof oligonucleotides by treatment with suitable reagents. For example,glass is prepared by treatment with silane reagents.

The present invention provides methods for determining whether amolecule of interest binds members of the oligonucleotide analoguearray. For instance, in one embodiment, a target molecule is hybridizedto the array and the resulting hybridization pattern is determined. Thetarget molecule includes genomic DNA, cDNA, unspliced RNA, mRNA, andrRNA, nucleic acid analogues, proteins and chemical polymers. The targetmolecules are optionally amplified prior to being hybridized to thearray, e.g., by PCR, LCR, or cloning methods.

The oligonucleotide analogue members of the array used in the abovemethods are synthesized by any described method for creating arrays. Inone embodiment, the oligonucleotide analogue members are attached to thesolid substrate, or synthesized on the solid substrate by light-directedvery large scale immobilized polymer synthesis, e.g., usingphoto-removable protecting groups during synthesis. In anotherembodiment, the oligonucleotide members are attached to the solidsubstrate by forming a plurality of channels adjacent to the surface ofsaid substrate, placing selected monomers in said channels to synthesizeoligonucleotide analogues at predetermined portions of selected regions,wherein the portion of the selected regions comprise oligonucleotideanalogues different from oligonucleotide analogues in at least one otherof the selected regions, and repeating the steps with the channelsformed along a second portion of the selected regions. The solidsubstrate is any suitable material as described above, including beads,slides, and arrays, each of which is constructed from, e.g., silica,polymers and glass.

DEFINITIONS

An “Oligonucleotide” is a nucleic acid sequence composed of two or morenucleotides. An oligonucleotide is optionally derived from naturalsources, but is often synthesized chemically. It is of any size. An“oligonucleotide analogue” refers to a polymer with two or moremonomeric subunits, wherein the subunits have some structural featuresin common with a naturally occurring oligonucleotide which allow it tohybridize with a naturally occurring oligonucleotide in solution. Forinstance, structural groups are optionally added to the ribose or baseof a nucleoside for incorporation into an oligonucleotide, such as amethyl or allyl group at the 2′-O position on the ribose, or a fluorogroup which substitutes for the 2′-O group, or a bromo group on theribonucleoside base. The phosphodiester linkage, or “sugar-phosphatebackbone” of the oligonucleotide analogue is substituted or modified,for instance with methyl phosphonates or O-methyl phosphates. Anotherexample of an oligonucleotide analogue for purposes of this disclosureincludes “peptide nucleic acids” in which native or modified nucleicacid bases are attached to a polyamide backbone. Oligonucleotideanalogues optionally comprise a mixture of naturally occurringnucleotides and nucleotide analogues. However, an oligonucleotide whichis made entirely of naturally occurring nucleotides (i.e., thosecomprising DNA or RNA), with the exception of a protecting group on theend of the oligonucleotide, such as a protecting group used duringstandard nucleic acid synthesis is not considered an oligonucleotideanalogue for purposes of this invention.

A “nucleoside” is a pentose glycoside in which the aglycone is aheterocyclic base; upon the addition of a phosphate group the compoundbecomes a nucleotide. The major biological nucleosides are β-glycosidederivatives of D-ribose or D-2-deoxyribose. Nucleotides are phosphateesters of nucleosides which are generally acidic in solution due to thehydroxy groups on the phosphate. The nucleosides of DNA and RNA areconnected together via phosphate units attached to the 3′ position ofone pentose and the 5′ position of the next pentose. Nucleotideanalogues and/or nucleoside analogues are molecules with structuralsimilarities to the naturally occurring nucleotides or nucleosides asdiscussed above in the context of oligonucleotide analogues.

A “nucleic acid reagent” utilized in standard automated oligonucleotidesynthesis typically caries a protected phosphate on the 3′ hydroxyl ofthe ribose. Thus, nucleic acid reagents are referred to as nucleotides,nucleotide reagents, nucleoside reagents, nucleoside phosphates,nucleoside-3′-phosphates, nucleoside phosphoramidites, phosphoramidites,nucleoside phosphonates, phosphonates and the like. It is generallyunderstood that nucleotide reagents carry a reactive, or activatible,phosphoryl or phosphonyl moiety in order to form a phosphodiesterlinkage.

A “protecting group” as used herein, refers to any of the groups whichare designed to block one reactive site in a molecule while a chemicalreaction is carried out at another reactive site. More particularly, theprotecting groups used herein are optionally any of those groupsdescribed in Greene, et al., Protective Groups In Organic Chemistry, 2ndEd., John Wiley & Sons, New York, N.Y., 1991, which is incorporatedherein by reference. The proper selection of protecting groups for aparticular synthesis is governed by the overall methods employed in thesynthesis. For example, in “light-directed” synthesis, discussed herein,the protecting groups are photolabile protecting groups such as NVOC,MeNPoc, and those disclosed in co-pending Application PCT/US93/10162(filed Oct. 22, 1993), incorporated herein by reference. In othermethods, protecting groups are removed by chemical methods and includegroups such as FMOC, DMT and others known to those of skill in the art.

A “purine” is a generic term based upon the specific compound “purine”having a skeletal structure derived from the fusion of a pyrimidine ringand an imidazole ring. It is generally, and herein, used to describe ageneric class of compounds which have an atom or a group of atoms addedto the parent purine compound, such as the bases found in the naturallyoccurring nucleic acids adenine (6-aminopurine) and guanine(2-amino-6-oxopurine), or less commonly occurring molecules such as2-amino-adenine, N⁶-methyladenine, or 2-methylguanine.

A “purine analogue” has a heterocyclic ring with structural similaritiesto a purine, in which an atom or group of atoms is substituted for anatom in the purine ring. For instance, in one embodiment, one or more Natoms of the purine heterocyclic ring are replaced by C atoms.

A “pyrimidine” is a compound with a specific heterocyclic diazine ringstructure, but is used generically by persons of skill and herein torefer to any compound having a 1,3-diazine ring with minor additions,such as the common nucleic acid bases cytosine, thymine, uracil,5-methylcytosine and 5-hydroxymethylcytosine, or the non-naturallyoccurring 5-bromo-uracil.

A “pyrimidine analogue” is a compound with structural similarity to apyrimidine, in which one or more atom in the pyrimidine ring issubstituted. For instance, in one embodiment, one or more of the N atomsof the ring are substituted with C atoms.

A “solid substrate” has fixed organizational support matrix, such assilica, polymeric materials, or glass. In some embodiments, at least onesurface of the substrate is partially planar. In other embodiments it isdesirable to physically separate regions of the substrate to delineatesynthetic regions, for example with trenches, grooves, wells or thelike. Example of solid substrates include slides, beads and arrays.

DESCRIPTION OF THE DRAWING

FIG. 1 shows two panels (FIG. 1A and FIG. 1B) illustrating thedifference in fluorescence intensity between matched and mismatchedprobes on an oligonucleotide analogue array.

FIG. 2 is a graphic illustration of specific light-directed chemicalcoupling of oligonucleotide analogue monomers to an array.

FIG. 3 shows the relative efficiency and specificity of hybridizationfor immobilized probe arrays containing adenine versus probe arrayscontaining 2,6-diaminopurine nucleotides. (3′-CATCGTAGAA-5′ (SEQ IDNO:1)).

FIG. 4 shows the effect of substituting adenine with 2,6-diaminopurine(D) in immobilized poly-dA probe arrays. (AAAAANAAAAA (SEQ ID NO:2)).

FIG. 5 shows the effects of substituting 5-propynyl-2′-deoxyuridine and2-amino-2′ deoxyadenosine in AT arrays on hybridization to a targetnucleic acid. (ATATAATATA (SEQ ID NO:3) and CGCGCCGCGC (SEQ ID NO:4)).

FIG. 6 shows the effects of dI and 7-deaza-dG substitutions inoligonucleotide arrays. (3′-ATGTT(G1G2G3G4G5)CGGGT-5′ (SEQ ID NO:5)).

DETAILED DESCRIPTION

Methods of synthesizing desired single stranded oligonucleotide andoligonucleotide analogue sequences are known to those of skill in theart. In particular, methods of synthesizing oligonucleotides andoligonucleotide analogues are found in, for example, OligonucleotideSynthesis: A Practical Approach, Gait, ed., IRL Press, Oxford (1984); W.H. A. Kuijpers Nucleic Acids Research 18(17), 5197 (1994); K. L. DueholmJ. Org. Chem. 59, 5767-5773 (1994), and S. Agrawal (ed.) Methods inMolecular Biology, volume 20, each of which is incorporated herein byreference in its entirety for all purposes. Synthesizing unimoleculardouble-stranded DNA in solution has also been described. See, copendingapplication Ser. No. 08/327,687, which is incorporated herein for allpurposes.

Improved methods of forming large arrays of oligonucleotides, peptidesand other polymer sequences with a minimal number of synthetic steps areknown. See, Pirrung et al, U.S. Pat. No. 5,143,854 (see also, PCTApplication No. WO 90/15070) and Fodor et al, PCT Publication No. WO92/10092, which are incorporated herein by reference, which disclosemethods of forming vast arrays of peptides, oligonucleotides and othermolecules using, for example, light-directed synthesis techniques. Seealso, Fodor et al, (1991) Science, 251, 767-77 which is incorporatedherein by reference for all purposes. These procedures for synthesis ofpolymer arrays are now referred to as VLSIPS™ procedures.

Using the VLSIPS™ approach, one heterogenous array of polymers isconverted, through simultaneous coupling at a number of reaction sites,into a different heterogenous array. See, U.S. application Ser. Nos.07/796,243 and 07/980,523, the disclosures of which are incorporatedherein for all purposes.

The development of VLSIPS™ technology as described in the above-notedU.S. Pat. No. 5,143,854 and PCT patent publication Nos. WO 90/15070 and92/10092 is considered pioneering technology in the fields ofcombinatorial synthesis and screening of combinatorial libraries. Morerecently, patent application Ser. No. 08/082,937, filed Jun. 25, 1993(incorporated herein by reference), describes methods for making arraysof oligonucleotide probes that are used to check or determine a partialor complete sequence of a target nucleic acid and to detect the presenceof a nucleic acid containing a specific oligonucleotide sequence.

Combinatorial Synthesis of Oligonucleotide Arrays

VLSIPS™ technology provides for the combinatorial synthesis ofoligonucleotide arrays. The combinatorial VLSIPS™ strategy allows forthe synthesis of arrays containing a large number of related probesusing a minimal number of synthetic steps. For instance, it is possibleto synthesize and attach all possible DNA 8mer oligonucleotides (4⁸, or65,536 possible combinations) using only 32 chemical synthetic steps. Ingeneral, VLSIPS™ procedures provide a method of producing 4^(n)different oligonucleotide probes on an array using only 4n syntheticsteps.

In brief, the light-directed combinatorial synthesis of oligonucleotidearrays on a glass surface proceeds using automated phosphoramiditechemistry and chip masking techniques. In one specific implementation, aglass surface is derivatized with a silane reagent containing afunctional group, e.g., a hydroxyl or amine group blocked by aphotolabile protecting group. Photolysis through a photolithographicmask is used selectively to expose functional groups which are thenready to react with incoming 5′-photoprotected nucleosidephosphoramidites. See, FIG. 2. The phosphoramidites react only withthose sites which are illuminated (and thus exposed by removal of thephotolabile blocking group). Thus, the phosphoramidites only add tothose areas selectively exposed from the preceding step. These steps arerepeated until the desired array of sequences have been synthesized onthe solid surface. Combinatorial synthesis of different oligonucleotideanalogues at different locations on the array is determined by thepattern of illumination during synthesis and the order of addition ofcoupling reagents.

In the event that an oligonucleotide analogue with a polyamide backboneis used in the VLSIPS™ procedure, it is generally inappropriate to usephosphoramidite chemistry to perform the synthetic steps, since themonomers do not attach to one another via a phosphate linkage. Instead,peptide synthetic method are substituted. See, e.g., Pirrung et al. U.S.Pat. No. 5,143,854.

Peptide nucleic acids are commercially available from, e.g., Biosearch,Inc. (Bedford, Mass.) which comprise a polyamide backbone and the basesfound in naturally occurring nucleosides. Peptide nucleic acids arecapable of binding to nucleic acids with high specificity, and areconsidered “oligonucleotide analogues” for purposes of this disclosure.Note that peptide nucleic acids optionally comprise bases other thanthose which are naturally occurring.

Hybridization of Nucleotide Analogues

The stability of duplexes formed between RNAs or DNAs are generally inthe order of RNA:RNA>RNA:DNA>DNA:DNA, in solution. Long probes havebetter duplex stability with a target, but poorer mismatchdiscrimination than shorter probes (mismatch discrimination refers tothe measured hybridization signal ratio between a perfect match probeand a single base mismatch probe. Shorter probes (e.g., 8-mers)discriminate mismatches very well, but the overall duplex stability islow. In order to optimize mismatch discrimination and duplex stability,the present invention provides a variety of nucleotide analoguesincorporated into polymers and attached in an array to a solidsubstrate.

Altering the thermal stability (T_(m)) of the duplex formed between thetarget and the probe using, e.g., known oligonucleotide analogues allowsfor optimization of duplex stability and mismatch discrimination. Oneuseful aspect of altering the T_(m), arises from the fact thatAdenine-Thymine (A-T) duplexes have a lower T_(m) than Guanine-Cytosine(G-C) duplexes, due in part to the fact that the A-T duplexes have 2hydrogen bonds per base-pair, while the G-C duplexes have 3 hydrogenbonds per base pair. In heterogeneous oligonucleotide arrays in whichthere is a non-uniform distribution of bases, it can be difficult tooptimize hybridization conditions for all probes simultaneously. Thus,in some embodiments, it is desirable to destabilize G-C-rich duplexesand/or to increase the stability of A-T-rich duplexes while maintainingthe sequence specificity of hybridization. This is accomplished, e.g.,by replacing one or more of the native nucleotides in the probe (or thetarget) with certain modified, non-standard nucleotides. Substitution ofguanine residues with 7-deazaguanine, for example, will generallydestabilize duplexes, whereas substituting adenine residues with2,6-diaminopurine will enhance duplex stability. A variety of othermodified bases are also incorporated into nucleic acids to enhance ordecrease overall duplex stability while maintaining specificity ofhybridization. The incorporation of 6-aza-pyrimidine analogs intooligonucleotide probes generally decreases their binding affinity forcomplementary nucleic acids. Many 5-substituted pyrimidinessubstantially increase the stability of hybrids in which they have beensubstituted in place of the native pyrimidines in the sequence. Examplesinclude 5-bromo-, 5-methyl-, 5-propynyl-, 5-(imidazol-2-yl)- and5-(thiazol-2-yl)-derivatives of cytosine and uracil.

Many modified nucleosides, nucleotides and various bases suitable forincorporation into nucleosides are commercially available from a varietyof manufacturers, including the SIGMA chemical company (Saint Louis,Mo.), R&D systems (Minneapolis, Minn.), Pharmacia LKB Biotechnology(Piscataway, N.J.), CLONTECH Laboratories, Inc. (Palo Alto, Calif.),Chem Genes Corp., Aldrich Chemical Company (Milwaukee, Wis.), GlenResearch, Inc., GIBCO BRL Life Technologies, Inc. (Gaithersberg, Md.),Fluka Chemica-Biochemika Analytika (Fluka Chemie AG, Buchs,Switzerland), Invitrogen, San Diego, Calif., and Applied Biosystems(Foster City, Calif.), as well as many other commercial sources known toone of skill. Methods of attaching bases to sugar moieties to formnucleosides are known. See, e.g., Lukevics and Zablocka (1991),Nucleoside Synthesis: Organosilicon Methods Ellis Horwood LimitedChichester, West Sussex, England and the references therein. Methods ofphosphorylating nucleosides to form nucleotides, and of incorporatingnucleotides into oligonucleotides are also known. See, e.g., Agrawal(ed) (1993) Protocols for Oligonucleotides and Analogues, Synthesis andProperties, Methods in Molecular Biology volume 20, Humana Press,Towota, N.J., and the references therein. See also, Crooke and Lebleu,and Sanghvi and Cook, and the references cited therein, both supra.

Groups are also linked to various positions on the nucleoside sugar ringor on the purine or pyrimidine rings which may stabilize the duplex byelectrostatic interactions with the negatively charged phosphatebackbone, or through hydrogen bonding interactions in the major andminor groves. For example, adenosine and guanosine nucleotides areoptionally substituted at the N² position with an imidazolyl propylgroup, increasing duplex stability. Universal base analogues such as3-nitropyrrole and 5-nitroindole are optionally included inoligonucleotide probes to improve duplex stability through base stackinginteractions.

Selecting the length of oligonucleotide probes is also an importantconsideration when optimizing hybridization specificity. In general,shorter probe sequences are more specific than longer ones, in that theoccurrence of a single-base mismatch has a greater destabilizing effecton the hybrid duplex. However, as the overall thermodynamic stability ofhybrids decreases with length, in some embodiments it is desirable toenhance duplex stability for short probes globally. Certainmodifications of the sugar moiety in oligonucleotides provide usefulstabilization, and these can be used to increase the affinity of probesfor complementary nucleic acid sequences. For example, 2′-O-methyl-,2′-O-propyl-, and 2′-O-allyl-oligoribonucleotides have higher bindingaffinities for complementary RNA sequences than their unmodifiedcounterparts. Probes comprised of 2′-fluoro-2′-deoxyollgoribonucleotidesalso form more stable hybrids with RNA than do their unmodifiedcounterparts.

Replacement or substitution of the internucleotide phosphodiesterlinkage in oligo- or poly-nucleotides is also used to either increase ordecrease the affinity of probe-target interactions. For example,substituting phosphodiester linkages with phosphorothioate orphosphorodithioate linkages generally lowers duplex stability, withoutaffecting sequence specificity. Substitutions with a non-ionicmethylphosphonate linkage (racemic, or preferably, Rp stereochemistry)have a stabilizing influence on hybrid formation. Neutral or cationicphosphoramidate linkages also result in enhanced duplex stabilization.The phosphate diester backbone has been replaced with a variety of otherstabilizing, non-natural linkages which have been studied as potentialantisense therapeutic agents. See, e.g., Crooke and Lebleu (eds) (1993)Antisense Research Applications CRC Press; and, Sanghvi and Cook (eds)(1994) Carbohydrate modifications in Antisense Research ACS Symp. Ser.#580 ACS, Washington D.C. Very stable hybrids are formed between nucleicacids and probes comprised of peptide nucleic acids, in which the entiresugar-phosphate backbone has been replaced with a polyamide structure.

Another important factor which sometimes affects the use ofoligonucleotide probe arrays is the nature of the target nucleic acid.Oligodeoxynucleotide probes can hybridize to DNA and RNA targets withdifferent affinity and specificity. For example, probe sequencescontaining long “runs” of consecutive deoxyadenosine residues form lessstable hybrids with complementary RNA sequences than with thecomplementary DNA sequences. Substitution of dA in the probe with either2,6-diaminopurine deoxyriboside, or 2′-alkoxy- or 2′-fluoro-dA enhanceshybridization with RNA targets.

Internal structure within nucleic acid probes or the targets alsoinfluences hybridization efficiency. For example, GC-rich sequences, andsequences containing “runs” of consecutive G residues frequentlyself-associate to form higher-order structures, and this can inhibittheir binding to complementary sequences. See, Zimmermann et al. (1975)J. Mol. Biol 92: 181; Kim (1991) Nature 351: 331; Sen and Gilbert (1988)Nature 335: 364; and Sunquist and Klug (1989) Nature 342: 825. Thesestructures are selectively destabilized by the substitution of one ormore guanine residues with one or more of the following purines orpurine analogs: 7-deazaguanine, 8-aza-7-deazaguanine, 2-aminopurine,1H-purine, and hypoxanthine, in order to enhance hybridization.

Modified nucleic acids and nucleic acid analogs can also be used toimprove the chemical stability of probe arrays. For example, certainprocesses and conditions that are useful for either the fabrication orsubsequent use of the arrays, may not be compatible with standardoligonucleotide chemistry, and alternate chemistry can be employed toovercome these problems. For example, exposure to acidic conditions willcause depurination of purine nucleotides, ultimately resulting in chaincleavage and overall degradation of the probe array. In this case,adenine and guanine are replaced with 7-deazaadenine and 7-deazaguanine,respectively, in order to stabilize the oligonucleotide probes towardsacidic conditions which are used during the manufacture or use of thearrays.

Base, phosphate and sugar modifications are used in combination to makehighly modified oligonucleotide analogues which take advantage of theproperties of each of the various modifications. For example,oligonucleotides which have higher binding affinities for complementarysequences than their unmodified counterparts (e.g., 2′-O-methyl-,2-O-propyl-, and 2′-O-allyl oligonucleotides) can be incorporated intooligonucleotides with modified bases (deazaguanine,8-aza-7-deazaguanine, 2-aminopurine, 1H-purine, hypoxanthine and thelike) with non-ionic methylphosphonate linkages or neutral or cationicphosphoramidate linkages, resulting in additive stabilization of duplexformation between the oligonucleotide and a target nucleic acid. Forinstance, one preferred oligonucleotide comprises a2′-O-methyl-2,6-diaminopurineriboside phosphorothioate. Similarly, anyof the modified bases described herein can be incorporated into peptidenucleic acids, in which the entire sugar-phosphate backbone has beenreplaced with a polyamide structure.

Thermal equilibrium studies, kinetic “on-rate” studies, and sequencespecificity analysis is optionally performed for any targetoligonucleotide and probe or probe analogue. The data obtained shows thebehavior of the analogues upon duplex formation with targetoligonucleotides. Altered duplex stability conferred by usingoligonucleotide analogue probes are ascertained by following, e.g.,fluorescence signal intensity of oligonucleotide analogue arrayshybridized with a target oligonucleotide over time. The data allowoptimization of specific hybridization conditions at, e.g., roomtemperature (for simplified diagnostic applications).

Another way of verifying altered duplex stability is by following thesignal intensity generated upon hybridization with time. Previousexperiments using DNA targets and DNA chips have shown that signalintensity increases with time, and that the more stable duplexesgenerate higher signal intensities faster than less stable duplexes. Thesignals reach a plateau or “saturate” after a certain amount of time dueto all of the binding sites becoming occupied. These data allow foroptimization of hybridization, and determination of equilibrationconditions at a specified temperature.

Graphs of signal intensity and base mismatch positions are plotted andthe ratios of perfect match versus mismatches calculated. Thiscalculation shows the sequence specific properties of nucleotideanalogues as probes. Perfect match/mismatch ratios greater than 4 areoften desirable in an oligonucleotide diagnostic assay because, for adiploid genome, ratios of 2 have to be distinguished (e.g., in the caseof a heterozygous trait or sequence).

Target Nucleic Acids which Comprise Nucleotide Analogues

Modified nucleotides and nucleotide analogues are incorporatedsynthetically or enzymatically into DNA or RNA target nucleic acids forhybridization analysis to oligonucleotide arrays. The incorporation ofnucleotide analogues in the target optimizes the hybridization of thetarget in terms of sequence specificity and/or the overall affinity ofbinding to oligonucleotide and oligonucleotide analogue probe arrays.The use of nucleotide analogues in either the oligonucleotide array orthe target nucleic acid, or both, improves optimizability ofhybridization interactions. Examples of useful nucleotide analogueswhich are substituted for naturally occurring nucleotides include7-deazaguanosine, 2,6-diaminopurine nucleotides, 5-propynyl and other5-substituted pyrimidine nucleotides, 2′-fluoro and2′-methoxy-2′-deoxynucleotides and the like.

These nucleotide analogues are incorporated into nucleic acids using thesynthetic methods described supra, or using DNA or RNA polymerases. Thenucleotide analogues are preferably incorporated into target nucleicacids using in vitro amplification methods such as PCR, LCR,Qβ-replicase expansion, in vitro transcription (e.g., nick translationor random-primer transcription) and the like. Alternatively, thenucleotide analogues are optionally incorporated into cloned nucleicacids by culturing a cell which comprises the cloned nucleic acid inmedia which includes a nucleotide analogue.

Similar to the use of nucleotide analogues in probe arrays,7-deazaguanosine is used in target nucleic acids to substitute for G/dGto enhance target hybridization by reducing secondary structure insequences containing runs of poly-G/dG. 6-diaminopurine nucleotide'ssubstitute for A/dA to enhance target hybridization through enhancedH-bonding to T or U rich probes. 5-propynyl and other 5-substitutedpyrimidine nucleotides substitute for natural pyrimidines to enhancetarget hybridization to certain purine rich probes. 2′-fluoro and2′-methoxy-2′-deoxynucleotides substitute for natural nucleotides toenhance target hybridization to similarly substituted probe sequences.

Synthesis of 5′-Photoprotected 2′-O Alkyl ribonucleotide analogues

The light-directed synthesis of complex arrays of nucleotide analogueson a glass surface is achieved by derivatizing cyanoethylphosphoramidite nucleotides and nucleotide analogues (e.g., nucleosideanalogues of uridine, thymidine, cytidine, adenosine and guanosine, withphosphates) with, for example, the photolabile MeNPoc group in the5′-hydroxyl position instead of the usual dimethoxytrityl group. See,application SN PCT/US94/12305.

Specific base-protected 2′-O alkyl nucleosides are commerciallyavailable, from, e.g., Chem Genes Corp. (MA). The photolabile MeNPocgroup is added to the 5′-hydroxyl position followed by phosphitylationto yield cyanoethyl phosphoramidite monomers. Commercially availablenucleosides are optionally modified (e.g., by 2-O-alkylation) to createnucleoside analogues which are used to generate oligonucleotideanalogues.

Modifications to the above procedures are used in some embodiments toavoid significant addition of MenPoc to the 3′-hydroxyl position. Forinstance, in one embodiment, a 2′-O-methyl ribonucleotide analogue isreacted with DMT-Cl {di(p-methoxyphenyl)phenylchloride} in the presenceof pyridine to generate a 2′-O-methyl-5′-O-DMT ribonucleotide analogue.This allows for the addition of TBDMS to the 3′-O of the ribonucleosideanalogue by reaction with TBDMS-Triflate(t-butyldimethylsilyltrifluoromethane-sulfonate) in the presence oftriethylamine in THF (tetrahydrofuran) to yield a2′-O-methyl-3′-O-TBDMS-5′-O-DMT ribonucleotide base analogue. Thisanalogue is treated with TCAA (trichloroacetic acid) to cleave off theDMT group, leaving a reactive hydroxyl group at the 5′ position. MeNPocis then added to the oxygen of the 5′ hydroxyl group using MenPoc-Cl inthe presence of pyridine. The TBDMS group is then cleaved withF.sup.-(e.g., NaF) to yield a ribonucleotide base analogue with a MeNPocgroup attached to the 5′ oxygen on the nucleotide analogue. Ifappropriate, this analogue is phosphitylated to yield a phosphoramiditefor oligonucleotide analogue synthesis. Other nucleosides or nucleosideanalogues are protected by similar procedures.

Synthesis of Oligonucleotide Analogue Arrays on Chips

Other than the use of photoremovable protecting groups, the nucleosidecoupling chemistry used in VLSIPS™ technology for synthesizingoligonucleotides and oligonucleotide analogues on chips is similar tothat used for oligonucleotide synthesis. The oligonucleotide istypically linked to the substrate via the 3′-hydroxyl group of theoligonucleotide and a functional group on the substrate which results inthe formation of an ether, ester, carbamate or phosphate ester linkage.Nucleotide or oligonucleotide analogues are attached to the solidsupport via carbon-carbon bonds using, for example, supports having(poly)trifluorochloroethylene surfaces, or preferably, by siloxane bonds(using, for example, glass or silicon oxide as the solid support).Siloxane bonds with the surface of the support are formed in oneembodiment via reactions of surface attaching portions bearingtrichlorosilyl or trialkoxysilyl groups. The surface attaching groupshave a site for attachment of the oligonucleotide analogue portion. Forexample, groups which are suitable for attachment include amines,hydroxyl, thiol, and carboxyl. Preferred surface attaching orderivatizing portions include aminoalkylsilanes and hydroxyalkylsilanes.In particularly preferred embodiments, the surface attaching portion ofthe oligonucleotide analogue is eitherbis(2-hydroxyethyl)-aminopropyltriethoxysilane,n-(3-triethoxysilylpropyl)-4-hydroxybutylamide,aminopropyltriethoxysilane or hydroxypropyltriethoxysilane.

The oligoribonucleotides generated by synthesis using ordinaryribonucleotides are usually base labile due to the presence of the2′-hydroxyl group. 2′-O-methyloligoribonucleotides (2′-OMeORNs),analogues of RNA where the 2′-hydroxyl group is methylated, are DNAseand RNAse resistant, making them less base labile. Sproat, B. S., andLamond, A. I. in Oligonucleotides and Analogues: A Practical Approach,edited by F. Eckstein, New York: IRL Press at Oxford University Press1991, pp. 49-86, incorporated herein by reference for all purposes, havereported the synthesis of mixed sequences of2′-O-Methoxy-oligoribonucleotides (2′-O-MeORNs) using dimethoxytritylphosphoramidite chemistry. These 2′-O-MeORNs display greater bindingaffinity for complementary nucleic acids than their unmodifiedcounterparts. Other embodiments of the invention provide mechanicalmeans to generate oligonucleotide analogues. These techniques arediscussed in co-pending application Ser. No. 07/796,243, filed Nov. 22,1991, which is incorporated herein by reference in its entirety for allpurposes. Essentially, oligonucleotide analogue reagents are directedover the surface of a substrate such that a predefined array ofoligonucleotide analogues is created. For instance, a series ofchannels, grooves, or spots are formed on or adjacent to a substrate.Reagents are selectively flowed through or deposited in the channels,grooves, or spots, forming an array having different oligonucleotidesand/or oligonucleotide analogues at selected locations on the substrate.

Detection of Hybridization

In one embodiment, hybridization is detected by labeling a target with,e.g., fluorescein or other known visualization agents and incubating thetarget with an array of oligonucleotide analogue probes. Upon duplexformation by the target with a probe in the array (or triplex formationin embodiments where the array comprises unimolecular double-strandedprobes), the fluorescein label is excited by, e.g., an argon laser anddetected by viewing the array, e.g., through a scanning confocalmicroscope.

Sequencing by Hybridization

Current sequencing methodologies are highly reliant on complexprocedures and require substantial manual effort. Conventional DNAsequencing technology is a laborious procedure requiring electrophoreticsize separation of labeled DNA fragments. An alternative approachinvolves a hybridization strategy carried out by attaching target DNA toa surface. The target is interrogated with a set of oligonucleotideprobes, one at a lime (see, application SN PCT/US94/12305).

A preferred method of oligonucleotide probe array synthesis involves theuse of light to direct the synthesis of oligonucleotide analogue probesin high-density, miniaturized arrays. Matrices of spatially-definedoligonucleotide analogue probe arrays were generated. The ability to usethese arrays to identify complementary sequences was demonstrated byhybridizing fluorescent labeled oligonucleotides to the matricesproduced.

Oligonucleotide analogue arrays are used, e.g., to study sequencespecific hybridization of nucleic acids, or protein-nucleic acidinteractions. Oligonucleotide analogue arrays are used to define thethermodynamic and kinetic rules governing the formation and stability ofoligonucleotide and oligonucleotide analogue complexes.

Oligonucleotide Analogue Probe Arrays and Libraries

The use of oligonucleotide analogues in probe arrays provides severalbenefits as compared to standard oligonucleotide arrays. For instance,as discussed supra, certain oligonucleotide analogues have enhancedhybridization characteristics to complementary nucleic acids as comparedwith oligonucleotides made of naturally occurring nucleotides. Oneprimary benefit of enhanced hybridization characteristics is thatoligonucleotide analogue probes are optionally shorter thancorresponding probes which do not include nucleotide analogues. Standardoligonucleotide probe arrays typically require fairly long probes (about15-25 nucleotides) to achieve strong binding to target nucleic acids.The use of such long probes is disadvantageous for two reasons. First,the longer the probe, the more synthetic steps must be performed to makethe probe and any probe array comprising the probe. This increases thecost of making the probes and arrays. Furthermore, as each syntheticstep results in less than 100% coupling for every nucleotide, thequality of the probes degrades as they become longer. Secondly, shortprobes provide better mis-match discrimination for hybridization to atarget nucleic acid. This is because a single base mismatch for a shortprobe-target hybridization is less destabilizing than a single mismatchfor a long probe-target hybridization. Thus, it is harder to distinguisha single probe-target mismatch when the probe is a 20-mer than when theprobe is an 8-mer. Accordingly, the use of short oligonucleotideanalogue probes reduces costs and increases mismatch discrimination inprobe arrays.

The enhanced hybridization characteristics of oligonucleotide analoguesalso allows for the creation of oligonucleotide analogue probe arrayswhere the probes in the arrays have substantial secondary structure. Forinstance, the oligonucleotide analogue probes are optionally configuredto be fully or partially double stranded on the array. The probes areoptionally complexed with complementary nucleic acids, or are optionallyunimolecular oligonucleotides with self-complementary regions. Librariesof diverse double-stranded oligonucleotide analogue probes are used, forexample, in screening studies to determine binding affinity of nucleicacid binding proteins, drugs, or oligonucleotides (e.g., to examinetriple helix formation). Specific oligonucleotide analogues are known tobe conducive to the formation of unusual secondary structure. See,Durland (1995) Bioconjugate Chem. 6: 278-282. General strategies forusing unimolecular double-stranded oligonucleotides as probes and forlibrary generation is described in application Ser. No. 08/327,687, andsimilar strategies are applicable to oligonucleotide analogue probes.

In general, a solid support, which optionally has an attached spacermolecule is attached to the distal end of the oligonucleotide analogueprobe. The probe is attached as a single unit, or synthesized on thesupport or spacer in a monomer by monomer approach using the VLSIPS™ ormechanical partitioning methods described supra. Where theoligonucleotide analogue arrays are fully double-stranded,oligonucleotides (or oligonucleotide analogues) complementary to theprobes on the array are hybridized to the array.

In some embodiments, molecules other than oligonucleotides, such asproteins, dyes, co-factors, linkers and the like are incorporated intothe oligonucleotide analogue probe, or attached to the distal end of theoligomer, e.g., as a spacing molecule, or as a probe or probe target.Flexible linkers are optionally used to separate complementary portionsof the oligonucleotide analogue.

The present invention also contemplates the preparation of libraries ofoligonucleotide analogues having bulges or loops in addition tocomplementary regions. Specific RNA bulges are often recognized byproteins (e.g., TAR RNA is recognized by the TAT protein of HIV).Accordingly, libraries of oligonucleotide analogue bulges or loops areuseful in a number of diagnostic applications. The bulge or loop can bepresent in the oligonucleotide analogue or linker portions.

Unimolecular analogue probes can be configured in a variety of ways. Inone embodiment, the unimolecular probes comprise linkers, for example,where the probe is arranged according to the formulaY-L.sup.¹-X¹-L²-X.sup², in which Y represents a solid support, X¹ and X²represent a pair of complementary oligonucleotides or oligonucleotideanalogues, L¹ represents a bond or a spacer, and L² represents a linkinggroup having sufficient length such that X¹ and X² form adouble-stranded oligonucleotide. The general synthetic andconformational strategy used in generating the double-strandedunimolecular probes is similar to that described in co-pendingapplication Ser. No. 08/327,687, except that any of the elements of theprobe (L¹, X¹, L² and X²) comprises a nucleotide or an oligonucleotideanalogue. For instance, in one embodiment X¹ is an oligonucleotideanalogue.

The oligonucleotide analogue probes are optionally arranged to present avariety of moieties. For example, structural components are optionallypresented from the middle of a conformationally restrictedoligonucleotide analogue probe. In these embodiments, the analogueprobes generally have the structure —X¹¹—Z—X¹² wherein X¹¹ and X¹² arecomplementary oligonucleotide analogues and Z is a structural elementpresented away from the surface of the probe array. Z can include anagonist or antagonist for a cell membrane receptor, a toxin, venom,viral epitope, hormone, peptide, enzyme, cofactor, drug, protein,antibody or the like.

General Tiling Strategies for Detection of a Polymorphism in a TargetOligonucleotide

In diagnostic applications, oligonucleotide analogue arrays (e.g.,arrays on chips, slides or beads) are used to determine whether thereare any differences between a reference sequence and a targetoligonucleotide, e.g., whether an individual has a mutation orpolymorphism in a known gene. As discussed supra, the oligonucleotidetarget is optionally a nucleic acid such as a PCR amplicon whichcomprises one or more nucleotide analogues. In one embodiment, arraysare designed to contain probes exhibiting complementarity to one or moreselected reference sequence whose sequence is known. The arrays are usedto read a target sequence comprising either the reference sequenceitself or variants of that sequence. Any polynucleotide of knownsequence is selected as a reference sequence. Reference sequences ofinterest include sequences known to include mutations or polymorphismsassociated with phenotypic changes having clinical significance in humanpatients. For example, the CFTR gene and P53 gene in humans have beenidentified as the location of several mutations resulting in cysticfibrosis or cancer respectively. Other reference sequences of interestinclude those that serve to identify pathogenic microorganisms and/orare the site of mutations by which such microorganisms acquire drugresistance (e.g., the HIV reverse transcriptase gene for HIVresistance). Other reference sequences of interest include regions wherepolymorphic variations are known to occur (e.g., the D-loop region ofmitochondrial DNA). These reference sequences also have utility for,e.g., forensic, cladistic, or epidemiological studies.

Other reference sequences of interest include those from the genome ofpathogenic viruses (e.g., hepatitis (A, B, or C), herpes virus (e.g.,VZV, HSV-1, HAV-6, HSV-II, CMV, and Epstein Barr virus), adenovirus,influenza virus, flaviviruses, echovirus, rhinovirus, coxsackie virus,cornovirus, respiratory syncytial virus, mumps virus, rotavirus, measlesvirus, rubella virus, parvovirus, vaccinia virus, HTLV virus, denguevirus, papillomavirus, molluscum virus, poliovirus, rabies virus, JCvirus and arboviral encephalitis virus. Other reference sequences ofinterest are from genomes or episomes of pathogenic bacteria,particularly regions that confer drug resistance or allow phylogeniccharacterization of the host (e.g., 16S rRNA or corresponding DNA). Forexample, such bacteria include chlamydia, rickettsial bacteria,mycobacteria, staphylococci, treptocci, pneumonococci, meningococci andconococci, klebsiella, proteus, serratia, pseudomonas, legionella,diphtheria, salmonella, bacilli, cholera, tetanus, botulism, anthrax,plague, leptospirosis, and Lymes disease bacteria. Other referencesequences of interest include those in which mutations result in thefollowing autosomal recessive disorders: sickle cell anemia,β-thalassemia, phenylketonuria, galactosemia, Wilson's disease,hemochromatosis, severe combined immunodeficiency, alpha-1-antitrypsindeficiency, albinism, alkaptonuria, lysosomal storage diseases andEhlers-Danlos syndrome. Other reference sequences of interest includethose in which mutations result in X-linked recessive disorders:hemophilia, glucose-6-phosphate dehydrogenase, agammaglobulimenia,diabetes insipidus, Lesch-Nyhan syndrome, muscular dystrophy,Wiskott-Aldrich syndrome, Fabry's disease and fragile X-syndrome. Otherreference sequences of interest includes those in which mutations resultin the following autosomal dominant disorders: familialhypercholesterolemia, polycystic kidney disease, Huntington's disease,hereditary spherocytosis, Marfan's syndrome, von Willebrand's disease,neurofibromatosis, tuberous sclerosis, hereditary hemorrhagictelangiectasia, familial colonic polyposis, Ehlers-Danlos syndrome,myotonic dystrophy, muscular dystrophy, osteogenesis imperfecta, acuteintermittent porphyria, and von Hippel-Lindau disease.

Although an array of oligonucleotide analogue probes is usually laiddown in rows and columns for simplified data processing, such a physicalarrangement of probes on the solid substrate is not essential. Providedthat the spatial location of each probe in an array is known, the datafrom the probes is collected and processed to yield the sequence of atarget irrespective of the physical arrangement of the probes on, e.g.,a chip. In processing the data, the hybridization signals from therespective probes is assembled into any conceptual array desired forsubsequent data reduction, whatever the physical arrangement of probeson the substrate. In one embodiment, a basic tiling strategy provides anarray of immobilized probes for analysis of a target oligonucleotideshowing a high degree of sequence similarity to one or more selectedreference oligonucleotide (e.g., detection of a point mutation in atarget sequence). For instance, a first probe set comprises a pluralityof probes exhibiting perfect complementarity with a selected referenceoligonucleotide. The perfect complementarity usually exists throughoutthe length of the probe. However, probes having a segment or segments ofperfect complementarity that is/are flanked by leading or trailingsequences lacking complementarity to the reference sequence can also beused. Within a segment of complementarity, each probe in the first probeset has at least one interrogation position that corresponds to anucleotide in the reference sequence. The interrogation position isaligned with the corresponding nucleotide in the reference sequence whenthe probe and reference sequence are aligned to maximize complementaritybetween the two. If a probe has more than one interrogation position,each corresponds with a respective nucleotide in the reference sequence.The identity of an interrogation position and corresponding nucleotidein a particular probe in the first probe set cannot be determined simplyby inspection of the probe in the first set. An interrogation positionand corresponding nucleotide is defined by the comparative structures ofprobes in the first probe set and corresponding probes from additionalprobe sets.

For each probe in the first set, there are, for purposes of the presentillustration, multiple corresponding probes from additional probe sets.For instance, there are optionally probes corresponding to eachnucleotide of interest in the reference sequence. Each of thecorresponding probes has an interrogation position aligned with thatnucleotide of interest. Usually, the probes from the additional probesets are identical to the corresponding probe from the first probe setwith one exception. The exception is that at the interrogation position,which occurs in the same position in each of the corresponding probesfrom the additional probe sets. This position is occupied by a differentnucleotide in the corresponding probe sets. Other tiling strategies arealso employed, depending on the information to be obtained.

The probes are oligonucleotide analogues which are capable ofhybridizing with a target nucleic sequence by complementarybase-pairing. Complementary base pairing includes sequence-specific basepairing, which comprises, e.g., Watson-Crick base pairing or other formsof base pairing such as Hoogsteen base pairing. The probes are attachedby any appropriate linkage to a support. 3′ attachment is more usual asthis orientation is compatible with the preferred chemistry used insolid phase synthesis of oligonucleotides and oligonucleotide analogues(with the exception of, e.g., analogues which do not have a phosphatebackbone, such as peptide nucleic acids).

EXAMPLES

The following examples are provided by way of illustration only and notby way of limitation. A variety of parameters can be changed or modifiedto yield essentially similar results.

One approach to enhancing oligonucleotide hybridization is to increasethe thermal stability (T_(m)) of the duplex formed between the targetand the probe using oligonucleotide analogues that are known to increaseT_(m)'s upon hybridization to DNA. Enhanced hybridization usingoligonucleotide analogues is described in the examples below, includingenhanced hybridization in oligonucleotide arrays.

Example 1 Solution Oligonucleotide Melting T_(m)

The T_(m) of 2′-O-methyl oligonucleotide analogues was compared to theT_(m) for the corresponding DNA and RNA sequences in solution. Inaddition, the T_(m) of 2′-O-methyl oligonucleotide:DNA, 2′-O-methyloligonucleotide:RNA and RNA:DNA duplexes in solution was alsodetermined. The T_(m) was determined by varying the sample temperatureand monitoring the absorbance of the sample solution at 260 nm. Theoligonucleotide samples were dissolved in a 0.1M NaCl solution with anoligonucleotide concentration of 2 μM. Table 1 summarizes the results ofthe experiment. The results show that the hybridization of DNA insolution has approximately the same T_(m) as the hybridization of DNAwith a 2′-O-methyl-substituted oligonucleotide analogue. The resultsalso show that the T_(m) for the T-O-methyl-substituted oligonucleotideduplex is higher than that for the correspondingRNA:2′-O-methyl-substituted oligonucleotide duplex, which is higher thanthe T_(m) for the corresponding DNA:DNA or RNA:DNA duplex.

TABLE 1 Solution Oligonucleotide Melting Experiments (+) =Target Sequence (5′-CTGAACGGTAGCATCTTGAC-3′) (SEQ ID NO: 6)* (−) =Complementary Sequence (5′GTCAAGATGCTACCGTTCAG-3′) (SEQ ID NO: 7)*Type of Oligonucleotide Type of Oligonucleotide Target Sequence (+)Complementary Sequence (+) T_(m)(° C.) DNA(+) DNA(-) 61.6 DNA(+)2′OMe(-) 58.6 2′OMe(+) DNA(-) 61.6 2′OMe(+) 2′OMe(-) 78.0 RNA(+) DNA(-)58.2 RNA(+) 2′OMe(-) 73.6 *T refers to thymine for the DNAoligonucletides, or uracil for the RNA oligonucleotides.

Example 2 Array Hybridization Experiments with DNA Chips andOligonucleotide Analogue Targets

A variable length DNA probe array on a chip was designed to discriminatesingle base mismatches in the 3 corresponding sequences5′-CTGAACGGTAGCATCTTGAC-3′ (SEQ ID NO:6) (DNA target),5′-CUGAACGGUAGCAUCUUGAC-3′ (SEQ ID NO:8) (RNA target) and5′-CUGAACGGUAGCAUCUUGAC-3′ (SEQ ID NO:9) (2′-O-methyl oligonucleotidetarget), and generated by the VLSIPS™ procedure. The Chip was designedwith adjacent 12-mers and 8-mers which overlapped with the 3 targetsequences as shown in Table 2.

TABLE 2 Array hybridization Experiments Target 1 (DNA) 8-mer probe(complement) 12-mer probe (complement)

Target 2 (RNA) 8-mer probe (complement) 12-mer probe (complement)

Target 3 (2′-O-Me oligo) 8-mer probe (complement) 12-mer probe(complement)

Target oligos were synthesized using standard techniques. The DNA and2′-O-methyl oligonucleotide analogue target oligonucleotides werehybridized to the chip at a concentration of 10 nM in 5×SSPE at 20° C.in sequential experiments. Intensity measurements were taken at eachprobe position in the 8-mer and 12-mer arrays over time. The rate ofincrease in intensity was then plotted for each probe position. The rateof increase in intensity was similar for both targets in the 8-mer probearrays, but the 12-mer probes hybridized more rapidly to the DNA targetoligonucleotide.

Plots of intensity versus probe position were generated for the RNA, DNAand 2-O-methyl oligonucleotides to ascertain mismatch discrimination.The 8-mer probes displayed similar mismatch discrimination against alltargets. The 12-mer probes displayed the highest mismatch discriminationfor the DNA targets, followed by the 2′-O-methyl target, with the RNAtarget showing the poorest mismatch discrimination.

Thermal equilibrium experiments were performed by hybridizing each ofthe targets to the chip for 90 minutes at 5° C. temperature intervals.The chip was hybridized with the target in 5×SSPE at a targetconcentration of 10 nM. Intensity measurements were taken at the end ofthe 90 minute hybridization at each temperature point as describedabove. All of the targets displayed similar stability, with minimalhybridization to the 8-mer probes at 30° C. In addition, all of thetargets showed similar stability in hybridizing to the 12-mer probes.Thus, the 2′-β-methyl oligonucleotide target had similar hybridizationcharacteristics to DNA and RNA targets when hybridized against DNAprobes.

Example 3 2′-O-Methyl-Substituted Oligonucleotide Chips

DMT-protected DNA and 2′-O-methyl phosphoramidites were used tosynthesize 8-mer probe arrays on a glass slide using the VLSIPS™ method.The resulting chip was hybridized to DNA and RNA targets in separateexperiments. The target sequence, the sequences of the probes on thechip and the general physical layout of the chip is described in Table3.

The chip was hybridized to the RNA and DNA targets in successiveexperiments. The hybridization conditions used were 10 nM target, in5×SSPE. The chip and solution were heated from 20° C. to 50° C., with afluorescence measurement taken at 5 degree intervals as described in SNPCT/US94/12305. The chip and solution were maintained at eachtemperature for 90 minutes prior to fluorescence measurements. Theresults of the experiment showed that DNA probes were equal or superiorto 2′-O-methyl oligonucleotide analogue probes for hybridization to aDNA target, but that the 2′-O-methyl analogue oligonucleotide probesshowed dramatically better hybridization to the RNA target than the DNAprobes. In addition, the 2′-O-methyl analogue oligonucleotide probesshowed superior mismatch discrimination of the RNA target compared tothe DNA probes. The difference in fluorescence intensity between thematched and mismatched analogue probes was greater than the differencebetween the matched and mismatched DNA probes, dramatically increasingthe signal-to-noise ratio. FIG. 1 displays the results graphically (FIG.1A). (M) and (P) indicate mismatched and perfectly matched probes,respectively. FIG. 1B illustrates the fluorescence intensity versuslocation on an example chip for the various probes at 20° C. using RNAand DNA targets.

TABLE 3 2′-O-methyl Oligonucleotide Analogues on a Chip.Target Sequence (DNA): 5′-CTGAACGGTAGCATCTTGAC-3′ (SEQ ID NO: 6)Target Sequence (RNA): 5′-CUGAACGGUAGCAUCUUGAC-3′ (SEQ ID NO: 8)Matching DNA 5′-CTTGCCAT oligonucleotide probe (SEQ ID NO: 10) {DNA (M)}Matching 2′-O-methyl 5′-CUUGCCAU oligonucleotide (SEQ ID NO: 11)analogue probe {2′OMe (M)} DNA oligonucleotide 5′-CTTGCTATprobe with 1 base (SEQ ID NO: 12) mismatch {DNA (P)} 2′-O-methyl oligo-5′-CUUGCUAU nucleotide analogue (SEQ ID NO: 13) probe with 1 basemismatch {2′OMe (M)}

Schematic Representation of 2′-O-Methyl/DNA Chip

Matching 2′-O-methyl oligonucleotide analogue probe 2′-O-methyloligonucleotide analogue probe with 1 base mismatch DNA oligonucleotideprobe with 1 base mismatch Matching DNA oligonucleotide probe

Example 4 Synthesis of Oligonucleotide Analogues

The reagent MeNPoc-Cl group reacts non-selectively with both the 5′ and3′ hydroxyls on 2′-O-methyl nucleoside analogues. Thus, to generate highyields of 5′-O-MeNPoc-2′-β-methylribonucleoside analogues for use inoligonucleotide analogue synthesis, the followingprotection-deprotection scheme was utilized.

The protective group DMT was added to the 5′-O position of the2′-β-methylribonucleoside analogue in the presence of pyridine. Theresulting 5′-O-DMT protected analogue was reacted with TBDMS-Triflate inTHF, resulting in the addition of the TBDMS group to the 3′-0 of theanalogue. The 5′-DMT group was then removed with TCAA to yield a free OHgroup at the 5′ position of the 2′-O-methyl ribonucleoside analogue,followed by the addition of MeNPoc-Cl in the presence of pyridine, toyield 5′-O-MeNPoc-3′-O-TBDMS-2′-O-methyl ribonucleoside analogue. TheTBDMS group was then removed by reaction with NaF, and the 3′-OH groupwas phosphitylated using standard techniques.

Two other potential strategies did not result in high specific yields of5′-O-MeNPoc-2′-O-methylribonucleoside. In the first, a less reactiveMeNPoc derivative was synthesized by reacting MeNPoc-Cl with N-hydroxysuccimide to yield MeNPoc-NHS. This less reactive photocleavable group(MeNPoc-NHS) was found to react exclusively with the 3′ hydroxyl on the2′-O-methylribonucleoside analogue. In the second strategy, an organotinprotection scheme was used. Dibutyltin oxide was reacted with the2′-O-methylribonucleoside analogue followed by reaction with MeNPoc.Both 5′-O-MeNPoc and 3′-O-MeNPoc 2′-O-methylribonucleoside analogueswere obtained.

Example 5 Hybridization to Mixed-Sequence Oligodeoxynucleotide ProbesSubstituted with 2-amino-2′-deoxyadenosine (D)

To test the effect of a 2-amino-2′-deoxyadenosine (D) substitution in aheterogeneous probe sequence, two 4×4 oligodeoxynucleotide arrays wereconstructed using VLSIPS™ methodology and 5′-O-MeNPOC-protecteddeoxynucleoside phosphoramidites. Each array was comprised of thefollowing set of probes based on the sequence (3′)-CATCGTAGAA-(5′) (SEQID NO:1):

(SEQ ID NO: 14) 1.. -(HEG)-(3′)-CATN.sub.1GTAGAA-(5′) (SEQ ID NO: 15) 2.-(HEG)-(3′)-CATCN.sub.2TAGAA-(5′) (SEQ ID NO: 16) 3.-(HEG)-(3′)-CATCGN.sub.3AGAA-(5′) (SEQ ID NO: 17) 4.-(HEG)-(3′)-CATCGTN.sub.4GAA-(5′)where HEG=hexaethyleneglycol linker, and N is either A, G, C or T, sothat probes are obtained which contain single mismatches introduced ateach of four central locations in the sequence. The first probe arraywas constructed with all natural bases. In the second array,2-amino-2′-deoxyadenosine (D) was used in place of adenosine (A). Botharrays were hybridized with a 5′-fluorescein-labeledoligodeoxynucleotide target, (5′)-Fl-d(CTGAACGGTAGCATCTTGAC)-(3′) (SEQID NO:18), which contained a sequence (in bold) complementary to thebase probe sequence. The hybridization conditions were: 10 nM target in5×SSPE buffer at 22° C. with agitation. After 30 minutes, the chip wasmounted on the flowcell of a scanning laser confocal fluorescencemicroscope, rinsed briefly with 5×SSPE buffer at 22° C., and then asurface fluorescence image was obtained.

The relative efficiency of hybridization of the target to thecomplementary and single-base mismatched probes was determined bycomparing the average bound surface fluorescence intensity in thoseregions of the of the array containing the individual probe sequences.The results (FIG. 3) show that a 2-amino-2′-deoxyadenosine (D)substitution in a heterogeneous probe sequence is a relatively neutralone, with little effect on either the signal intensity or thespecificity of DNA-DNA hybridization, under conditions where the targetis in excess and the probes are saturated.

Example 6 Hybridization to a dA-Homopolymer Oligodeoxynucleotide ProbeSubstituted with 2-amino-2′-deoxyadenosine (D)

The following experiment was performed to compare the hybridization of2′-deoxyadenosine containing homopolymer arrays with2-amino-2′-deoxyadenosine homopolymer arrays. The experiment wasperformed on two 11-mer oligodeoxynucleotide probe containing arrays.Two 11-mer oligodeoxynucleotide probe sequences were synthesized on achip using 5′-O-MeNPOC-protected nucleoside phosphoramidites andstandard VLSIPS™ methodology.

The sequence of the first probe was: (HEG)-(3′)-d(AAAAANAAAAA)-(5′) (SEQID NO:19); where HEG=hexaethyleneglycol linker, and N is either A, G, Cor T. The second probe was the same, except that dA was replaced by2-amino-2′-deoxyadenosine (D). The chip was hybridized with a5′-fluorescein-labeled oligodeoxynucleotide target,(5′)-Fl-d(TTTTTGTTTTT)-(3′) (SEQ ID NO:20), which contained a sequencecomplementary to the probe sequences where N═C. Hybridization conditionswere 10 nM target in 5×SSPE buffer at 22° C. with agitation. After 15minutes, the chip was mounted on the flowcell of a scanning laserconfocal fluorescence microscope, rinsed briefly with 5×SSPE buffer at22° C. (low stringency), and a surface fluorescence image was obtained.Hybridization to the chip was continued for another 5 hours, and asurface fluorescence image was acquired again. Finally, the chip waswashed briefly with 0.5×SSPE (high-stringency), then with 5×SSPE, andre-scanned.

The relative efficiency of hybridization of the target to thecomplementary and single-base mismatched probes was determined bycomparing the average bound surface fluorescence intensity in thoseregions of the of the array containing the individual probe sequences.The results (FIG. 4) indicate that substituting 2′-deoxyadenosine with2-amino-2′-deoxyadenosine in a d(A)_(n) homopolymer probe sequenceresults in a significant enhancement in specific hybridization to acomplementary oligodeoxynucleotide sequence.

Example 7 Hybridization to Alternating A-T Oligodeoxynucleotide ProbesSubstituted with 5-propynyl-2′-deoxyuridine (P) and2-amino-2′-deoxyadenosine (D)

Commercially available 5′-DMT-protected2′-deoxynucleoside/nucleoside-analog phosphoramidites (Glen Research)were used to synthesize two decanucleotide probe sequences on separateareas on a chip using a modified VLSIPS™ procedure. In this procedure, aglass substrate is initially modified with a terminal-MeNPOC-protectedhexaethyleneglycol linker. The substrate was exposed to light through amask to remove the protecting group from the linker in a checkerboardpattern. The first probe sequence was then synthesized in the exposedregion using DMT-phosphoramidites with acid-deprotection cycles, and thesequence was finally capped with (MeO)₂PNiPr₂/tetrazole followed byoxidation. A second checkerboard exposure in a different (previouslyunexposed) region of the chip was then performed, and the second probesequence was synthesized by the same procedure. The sequence of thefirst “control” probe was: -(HEG)-(3′)-CGCGCCGCGC-(5′) (SEQ ID NO:21);and the sequence of the second probe was one of the following:

1. -(HEG)-(3′)-d(ATATAATATA)-(5′) (SEQ ID NO: 22) 2.-(HEG)-(3′)-d(APAPAAPAPA)-(5′) (SEQ ID NO: 23) 3.-(HEG)-(3′)-d(DTDTDDTDTD)-(5′) (SEQ ID NO: 24) 4.-(HEG)-(3′)-d(DPDPDDPDPD)-(5′) (SEQ ID NO: 25)where HEG=hexaethyleneglycol linker, A=2′-deoxyadenosine, T=thymidine,D=2-amino-2′-deoxyadenosine, and P=5-propynyl-2′-deoxyuridine. Each chipwas then hybridized in a solution of a fluorescein-labeledoligodeoxynucleotide target,(5′)-Fluorescein-d(TATATTATAT)-(HEG)-d(GCGCGGCGCG)-(3′) (SEQ ID NO:26and SEQ ID NO:27), which is complementary to both the A/T and G/Cprobes. The hybridization conditions were: 10 nM target in 5×SSPE bufferat 22° C. with gentle shaking. After 3 hours, the chip was mounted onthe flowcell of a scanning laser confocal fluorescence microscope,rinsed briefly with 5×SSPE buffer at 22° C., and then a surfacefluorescence image was obtained. Hybridization to the chip was continuedovernight (total hybridization time=20 hr), and a surface fluorescenceimage was acquired again.

The relative efficiency of hybridization of the target to the A/T andsubstituted A/T probes was determined by comparing the average surfacefluorescence intensity bound to those parts of the chip containing theA/T or substituted probe to the fluorescence intensity bound to the G/Ccontrol probe sequence. The results (FIG. 5) show that 5-propynyl-dU and2-amino-dA substitution in an A/T-rich probe significantly enhances theaffinity of an oligonucleotide analogue for complementary targetsequences. The unsubstituted A/T-probe bound only 20% as much target asthe all-G/C-probe of the same length, while the D- & P-substituted A/Tprobe bound nearly as much (90%) as the G/C-probe. Moreover, thekinetics of hybridization are such that, at early times, the amount oftarget bound to the substituted A/T probes exceeds that which is boundto the all-G/C probe.

Example 8 Hybridization to Oligodeoxynucleotide Probes Substituted with7-deaza-2′-deoxyguanosine (ddG) and 2′-deoxyinosine (dI)

A 16×64 oligonucleotide array was constructed using VLSIPS™ methodology,with 5′-O-MeNPOC-protected nucleoside phosphoramidites, including theanalogs ddG, and dI. The array was comprised of the set of probesrepresented by the following sequence:

-(linker)-(3′)-d(A T G T T G₁ G₂ G₃G₄ G₅ C G G T)-(5′); (SEQ ID NO:28)where underlined bases are fixed, and the five internal deoxyguanosines(G₁₋₅) are substituted with G, ddG, dI, and T in all possible (1024total) combinations. A complementary oligonucleotide target, labeledwith fluorescein at the 5′-end:(5′)-Fl-d(C A A T A C A A C C C C C G C C C A T C C)-(3′) (SEQ IDNO:29), was hybridized to the array. The hybridization conditions were:5 nM target in 6×SSPE buffer at 22° C. with shaking. After 30 minutes,the chip was mounted on the flowcell of an Affymetrix scanning laserconfocal fluorescence microscope, rinsed once with 0.25×SSPE buffer at22° C., and then a surface fluorescence image was acquired.

The “efficiency” of target hybridization to each probe in the array isproportional to the bound surface fluorescence intensity in the regionof the chip where the probe was synthesized. The relative values for asubset of probes (those containing dG->ddG and dG->dI substitutionsonly) are shown in FIG. 6. Substitution of guanosine with7-deazaguanosine within the internal run of five G's results in asignificant enhancement in the fluorescence signal intensity whichmeasures hybridization. Deoxyinosine substitutions also enhancehybridization to the probe, but to a lesser extent. In this example, thebest overall enhancement is realized when the dG “run” is ˜40-60%substituted with 7-deaza-dG, with the substitutions distributed evenlythroughout the run (i.e., alternating dG/deaza-dG).

Example 9 Synthesis of5′-MeNPOC-2′-deoxyinosine-3′-(N,N-diisopropyl-2-cyanoethyl)phosphoramidite

2′-deoxyinosine (5.0 g, 20 mmole) was dissolved in 50 ml of dry DMF, and100 ml dry pyridine was added and evaporated three times to dry thesolution. Another 50 ml pyridine was added, the solution was cooled to−20° C. under argon, and 13.8 g (50 mmole) of MeNPOC-chloride in 20 mldry DCM was then added dropwise with stirring over 60 minutes. After 60minutes, the cold bath was removed, and the solution was allowed to stirovernight at room temperature. Pyridine and DCM were removed byevaporation, 500 ml of ethyl acetate was added, and the solution waswashed twice with water and then with brine (200 ml each). The aqueouswashes were combined and back-extracted twice with ethyl acetate, andthen all of the organic layers were combined, dried with Na₂SO₄, andevaporated under vacuum. The product was recrystallized from DCM toobtain 5.0 g (50% yield) of pure 5′-O-MeNPOC-2′-deoxyinosine as a yellowsolid (99% purity, according to ¹H-NMR and HPLC analysis).

The MeNPOC-nucleoside (2.5 g, 5.1 mmole) was suspended in 60 ml of dryCH₃CN and phosphitylated with2-cyanoethyl-N,N,N′,N′-tetraisopropylphosphorodiamidite (1.65 g/1.66 ml;5.5 mmole) and 0.47 g (2.7 mmole) of diisopropylammonium tetrazolide,according to the published procedure of Barone, et al. (Nucleic AcidsRes. (1984) 12, 4051-61). The crude phosphoramidite was purified byflash chromatography on silica gel (90:8:2 DCM-MeOH—Et₃N), co-evaporatedtwice with anhydrous acetonitrile and dried under vacuum for ˜24 hoursto obtain 2.8 g (80%) of the pure product as a yellow solid (98% purityas determined by ¹H/³¹P-NMR and HPLC).

Example 10 Synthesis of5′-MeNPOC-7-deaza-2′-deoxy(N-2-isobutyryl)-guanosine-3′-(N,N-diisopropyl-2-cyanoethyl)phosphoramidite

The protected nucleoside 7-deaza-2′-deoxy(N2-isobutyryl)guanosine (1.0g, 3 mmole; Chemgenes Corp., Waltham, Mass.) was dried by co-evaporatingthree times with 5 ml anhydrous pyridine and dissolved in 5 ml of drypyridine-DCM (75:25 by vol.). The solution was cooled to −45° C. (dryice/CH₃CN) under argon, and a solution of 0.9 g (3.3 mmole) MeNPOC-Cl in2 ml dry DCM was then added dropwise with stirring. After 30 minutes,the cold bath was removed, and the solution allowed to stir overnight atroom temperature. The solvents were evaporated, and the crude materialwas purified by flash chromatography on silica gel (2.5%-5% MeOH in DCM)to yield 1.5 g (88% yield)5′-MeNPOC-7-deaza-2′-deoxy(N-2-isobutyryl)guanosine as a yellow foam.The product was 98% pure according to ¹H-NMR and HPLC analysis.

The MeNPOC-nucleoside (1.25 g, 2.2 mmole) was phosphitylated accordingto the published procedure of Barone, et al. (Nucleic Acids Res. (1984)12, 4051-61). The crude product was purified by flash chromatography onsilica gel (60:35:5 hexane-ethyl acetate-Et.sub.3N), co-evaporated twicewith anhydrous acetonitrile and dried under vacuum for ˜24 hours toobtain 1.3 g (75%) of the pure product as a yellow solid (98% purity asdetermined by ¹H/³¹P-NMR and HPLC).

Example 11 Synthesis of5′-MeNPOC-2,6-bis(phenoxyacetyl)-2,6-diaminopurine-2′-deoxyriboside-3′-(N—,N-diisopropyl-2-cyanoethyl)phosphoramidite

The protected nucleoside2,6-bis(phenoxyacetyl)-2,6-diaminopurine-2′-deoxyriboside (8 mmole, 4.2g) was dried by coevaporating twice from anhydrous pyridine, dissolvedin 2:1 pyridine/DCM (17.6 ml) and then cooled to −40° C. MeNPOC-chloride(8 mmole, 2.18 g) was dissolved in DCM (6.6 mls) and added to reactionmixture dropwise. The reaction was allowed to stir overnight with slowwarming to room temperature. After the overnight stirring, another 2mmole (0.6 g) in DCM (1.6 ml) was added to the reaction at −40° C. andstirred for an additional 6 hours or until no unreacted nucleoside waspresent. The reaction mixture was evaporated to dryness, and the residuewas dissolved in ethyl acetate and washed with water twice, followed bya wash with saturated sodium chloride. The organic layer was dried withMgSO₄, and evaporated to a yellow solid which was purified by flashchromatography in DCM employing a methanol gradient to elute the desiredproduct in 51% yield.

The 5′-MeNPOC-nucleoside (4.5 mmole, 3.5 g) was phosphitylated accordingto the published procedure of Barone, et al. (Nucleic Acids Res. (1984)12, 4051-61). The crude product was purified by flash chromatography onsilica gel (99:0.5:0.5 DCM-MeOH—Et₃N). The pooled fractions wereevaporated to an oil, redissolved in a minimum amount of DCM,precipitated by the addition of 800 ml ice cold hexane, filtered, andthen dried under vacuum for ˜24 hours.

Overall yield was 56%, at greater than 96% purity by HPLC and¹H/³¹P-NMR.

Example 12 5′-O-MeNPOC-Protected Phosphoramidites for Incorporating7-deaza-2′ deoxyguanosine and 2′-deoxyinosine into VLSSIPS™Oligonucleotide Arrays

VLSIPS oligonucleotide probe arrays in which all or a subset of allguanosine residues are substitutes with 7-deaza-2′-deoxyguanosine and/or2′-deoxyinosine are highly desirable. This is because guanine-richregions of nucleic acids associate to form multi-stranded structures.For example, short tracts of G residues in RNA and DNA commonlyassociate to form tetrameric structures (Zimmermann et al. (1975) J.Mol. Biol. 92: 181; Kim, J. (1991) Nature 351: 331; Sen et al. (1988)Nature 335: 364; and Sunquist et al. (1989) Nature 342: 825). Theproblem this poses to chip hybridization-based assays is that suchstructures may compete or interfere with normal hybridization betweencomplementary nucleic acid sequences. However, by substituting the7-deaza-G analog into G-rich nucleic acid sequences, particularly at oneor more positions within a run of G residues, the tendency for suchprobes to form higher-order structures is suppressed, while maintainingessentially the same affinity and sequence specificity indouble-stranded structures. This has been exploited in order to reduceband compression in sequencing gels (Mizusawa, et al. (1986) N.A.R. 14:1319) to improve target hybridization to G-rich probe sequences inVLSIPS arrays. Similar results are achieved using inosine (see also,Sanger et al. (1977) P.N.A.S. 74: 5463).

For facile incorporation of 7-deaza-2′-deoxyguanosine and2′-deoxyinosine into oligonucleotide arrays using VLSIPS™ methods, anucleoside phosphoramidite comprising the analogue base which has a5′-O′-MeNPOC-protecting group is constructed. This building block wasprepared from commercially available nucleosides according to Scheme 1.These amidites pass the usual tests for coupling efficiency andphotolysis rate.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, modifications can be made thereto without departing fromthe spirit or scope of the appended claims.

All publications and patent applications cited in this application areherein incorporated by reference for all purposes as if each individualpublication or patent application were specifically and individuallyindicated to be incorporated by reference.

1. A composition comprising an array of oligonucleotide analoguesattached to a solid substrate.
 2. The composition of claim 1, whereinsaid solid substrate is selected from the group consisting of beads,chips, and slides.
 3. The composition of claim 1, wherein saidcomposition comprises an array of oligonucleotide analogue probes 5 to20 nucleotides in length.
 4. The composition of claim 1, wherein saidarray of oligonucleotide analogues comprises a nucleoside analogue withthe formula

wherein: R¹ is selected from the group consisting of hydrogen, methyl,hydroxy, alkoxy, alkylthio, halogen, cyano, and azido; R² is selectedfrom the group consisting of hydrogen, methyl, hydroxy, alkoxy,alkylthio, halogen, cyano, and azido; and Y is a heterocyclic moiety. 5.The composition of claim 1, wherein said array of oligonucleotideanalogues comprises a nucleoside analogue with the formula

wherein: R¹ is selected from the group consisting of hydrogen, hydroxyl,methyl, methoxy, ethoxy, propoxy, allyloxy, propargyloxy, Fluorine,Chlorine, and Bromine; R² is selected from the group consisting ofhydrogen, hydroxyl, methyl, methoxy, ethoxy, propoxy, allyloxy,propargyloxy, Fluorine, Chlorine, and Bromine; and Y is a base selectedfrom the group consisting of purines, purine analogues, pyrimidines,pyrimidine analogues, 3-nitropyrrole and 5-nitroindole.
 6. Thecomposition of claim 1, wherein said array of oligonucleotide analoguescomprises a peptide nucleic acid.
 7. The composition of claim 1, whereinsaid array of oligonucleotide analogues is resistant to RNAase A.
 8. Thecomposition of claim 1, wherein said solid substrate is attached to over1000 different oligonucleotide analogues.
 9. The composition of claim 1,wherein said array of oligonucleotide analogues comprises 2′-O-methylnucleotides.
 10. The composition of claim 1, wherein said array ofoligonucleotide analogues and said solid substrate comprises a pluralityof different members, each member having the formula:Y-L¹-X¹-L²-X² wherein, Y is a solid substrate; X¹ and X² arecomplementary oligonucleotides containing at least one nucleotideanalogue; L¹ is a spacer; L² is a linking group having sufficient lengthsuch that X¹ and X² form a double-stranded oligonucleotide.
 11. Thecomposition of claim 10, wherein said composition comprises a library ofunimolecular double-stranded oligonucleotide analogues.
 12. Thecomposition of claim 1, wherein said array of oligonucleotide analoguescomprises a conformationally restricted array of oligonucleotideanalogues with the formula:—X¹¹—Z—X¹² wherein X¹¹ and X¹² are complementary oligonucleotides oroligonucleotide analogues and Z is a presented moiety.
 13. Thecomposition of claim 1, wherein said array of oligonucleotide analoguescomprises a nucleotide with a base selected from the group of basesconsisting of 7-deazaguanosine, 2-aminopurine, 8-aza-7-deazaguanosine,1H-purine, and hypoxanthine.
 14. A composition comprising anoligonucleotide analogue array synthesized on a solid substrate, whereinsaid synthesis is performed by light-directed chemical coupling.
 15. Thecomposition of claim 14, wherein said solid substrate is derivitizedwith a silane reagent prior to synthesis of said oligonucleotideanalogue.
 16. A composition comprising an oligonucleotide analogue arraysynthesized on a solid substrate, wherein said synthesis is performed byflowing oligonucleotide analogue reagents over predetermined regions ofthe solid substrate.
 17. The composition of claim 16, wherein said solidsubstrate is derivitized with a silane reagent prior to synthesis ofsaid oligonucleotide analogue.
 18. A method of improving thehybridization of a nucleic acid to an oligonucleotide array, comprisingincorporating a base selected from the group consisting of7-deazaguanosine, 2-aminopurine, 8-aza-7-deazaguanosine, 1H-purine, andhypoxanthine into the oligonucleotides of the array.
 19. The method ofclaim 18, wherein the oligonucleotide is a homopolymer.
 20. A method ofdetermining if a target molecule is complementary to a probe, comprisingthe steps of: (a) synthesizing an oligonucleotide analogue array on asolid substrate; (b) exposing said oligonucleotide analogue array onsaid solid substrate to a target nucleic acid; and (c) determiningwhether an oligonucleotide analogue member of said oligonucleotideanalogue array binds to said target oligonucleotide.
 21. The method ofclaim 20, wherein said target nucleic acid is selected from the groupcomprising genomic DNA, cDNA, unspliced RNA, mRNA, and rRNA.
 22. Themethod of claim 20, wherein said target nucleic acid is amplified priorto said hybridization step.
 23. The method of claim 20, wherein saidoligonucleotide analogue array is synthesized on said solid support bylight-directed very large scale immobilized polymer synthesis.
 24. Themethod of claim 20, wherein said oligonucleotide analogue array issynthesized on said solid support by causing oligonucleotide analoguesynthetic reagents to flow over selected regions of said solid support.25. The method of claim 20, wherein said step (a) comprises the stepsof: i) forming a plurality of channels adjacent to the surface of saidsubstrate; ii) placing selected monomers in said channels to synthesizeoligonucleotide analogues at predetermined portions of selected regions,said portion of said selected regions comprising oligonucleotideanalogues different from oligonucleotide analogues in at least one otherof said selected regions; and iii) repeating steps i) and ii) with saidchannels formed along a second portion of said selected regions, therebyforming an array of oligonucleotide analogues.
 26. The method of claim20, wherein said solid substrate is selected from the group consistingof beads, slides, and chips.
 27. The method of claim 20, wherein saidsolid substrate is comprised of materials selected from the groupconsisting of silica, polymers and glass.
 28. The method of claim 20,wherein the oligonucleotide analogue members of said array aresynthesized using photoremovable protecting groups.
 29. The method ofclaim 20, wherein said oligonucleotide analogue array is synthesized bya process comprising selective incorporation of MeNPoc onto the 5′hydroxyl of a nucleoside analogue, which nucleoside analogue isincorporated into said oligonucleotide analogue array.
 30. The method ofclaim 20, wherein said oligonucleotide analogue array is synthesizedfrom phosphoramidite nucleoside reagents.
 31. A composition comprisingan array of oligonucleotide probes hybridized to a target nucleic acid,which target nucleic acid comprises a nucleotide analogue.
 32. Thecomposition of claim 31, wherein the target nucleic acid is a PCRamplicon.
 33. The composition of claim 31, wherein the oligonucleotideprobes comprise oligonucleotide analogues.
 34. The composition of claim31, wherein the target nucleic acid is an RNA nucleic acid.
 35. A methodof detecting a target nucleic acid, comprising enzymatically copying thetarget nucleic acid using nucleotides which comprise a nucleotideanalogue, thereby producing a nucleic acid analogue amplicon, andhybridizing the nucleic acid amplicon to an oligonucleotide array. 36.The method of claim 35, wherein the oligonucleotide array comprises anoligonucleotide analogue probe which is complementary to the nucleicacid analogue amplicon.